Methods of making glucose isomerase and of converting glucose to fructose

ABSTRACT

METHODS FOR THE CONVERSION OF D-GLUCOSE TO D-FRUCTOSE ARE DISCLOSED. ENZYMES DERIVED FROM AEROBACTER LEVANICUM ARE USED. A GLUCOSE ISOMERIZING ENZYME IS PRODUCED WHEN AEROBACTER LEVANICUM IS GROWN ON A SUBSTRATE CONTAINING XYLOSE, PREFERABLY UNPURIFIED BIRCHWOOD SULFITE LIQUOR.

United States Patent Int. Cl. C07g 7/02 US. Cl. 195-66 R 8 ClaimsABSTRACT OF THE DISCLOSURE Methods for the conversion of D-glucose toD-fructose are disclosed. Enzymes derived from Aerobacter levanicum areused. A glucose isomerizing enzyme is produced when Aerqbacterlevanz'cum is grown on a substrate containing xylose, preferablyunpurified birchwood sulfite liquor.

BACKGROUND OF THE INVENTION The commercial production of fructose fromglucose is desirable because of increased sweetness of the resultingproduct over pure glucose. The fully converted product is a syrup thatresembles invert sugar, i.e. it is about 50% fructose and 50% glucose.

The conversion of glucose to fructose can be catalyzed in general bybases, such as sodium hydroxide, but the conditions are such thatundesirable side products may result. Reports abound in the literature,both general and patent, concerning enzymes derived from microbiologicalsources which catalyze the conversion of glucose to fructose with aminimum of side reactions. All of the commercially feasible enzymesources in the past have been derived from fermentations of variousstrains of Streptomyces in a medium in which xylose or xylan is used toinduce the enzyme. A major disadvantage of such derived enzymes is thenecessity for a portion of the carbon source of the fermentation mediumto be composed of purified xylose. We have devised a method thatemploys, without any purification, hardwood sulfite liquors(particularly birchwood sulfite liquor) a readily available wasteproduct of pulp and paper mills, as the source of xylose. Themicroorganism Aerobacter levanicum is suitable for this purpose. Cellsof Aerobacter levanicum contain high amounts of glucose isomerizingactivity when grown on a source of xylose.

The assay of the glucose isomerizing activity of enzyme extracts iscarried out in 1 M glucose, 3 10- M CoCl 3X10 M MgSO 0.035 M phosphatebuffer at pH 7.5 and 55 C. The initial rate of enzymatic conversion ofglucose to fructose is determined by measuring the rate of fructoseformation by the HCl-skatole method, as described by Pogell in J. Biol.Chem. 211:143, 1954. One unit of activity corresponds to a rate oflaM/min. of fructose formed under the above conditions. The activity ofglucose isomerase obtained from simultaneous cell propagation and enzymeinduction is measured by using intact cells as enzyme instead of using acell-free extract.

SUMMARY OF THE INVENTION The invention comprises a method of isomerizingglucose to fructose using enzymes derived from the microorganism,Aerolbacter levanicum, grown on a suitable substrate containing xylose,a nitrogen source, and Co 3,813,320 Patented May 28, 1974 DETAILEDDESCRIPTION The microorganism used in this process is Aerobacterlevanicum obtained from the Northern Regional Research Laboratory of theUnited States Department of Agriculture at Peoria, Ill., and identifiedas deposit NRRL B-1678.

The A. levanicum organism can be grown on a medium containing carbon andnitrogen. In order to produce converting enzyme, however, xylose must bepresent in the substrate.

For isomerization to take place, Co must be present in the isomerizingmedium or the cells or enzyme must have been grown on a substratecontaining Co++. For best results -K+ or Mg++ also are present, eitherduring cell growth or in the isomerizing medium.

As previously mentioned, hardwood sulfite liquor, specifically birchwoodsulfite liquid is a suitable source of xylose. The birchwood sulfiteliquor (B.S.L.) is not purified or sterilized as is required in thegrowth of other microorganisms which produce glucose isomerase. This isan important commercial consideration as this syrup (the isomerizedglucose-fructose syrup) is intended to replace conventional invert sugarin certain syrups, candy and baking applications, and thus must becompetitive in price.

In producing the enzyme, the A. levanicum cells can be propagated on asuitable growth medium and then added to a xylose medium (B.S.L.) toinduce enzyme or the growth medium can contain xylose so that enzyme isinduced as the cells are grown.

The enzyme can be added to the glucose substrate in the form of airdried cells or the enzyme can be separated from the cells bysonification and only the enzyme added to the glucose substrate.Examples of both of these processes are given hereinafter.

A suitable medium for producing A. levanicum cells contains yeastextract or corn steep liquor as nitrogen sources and D-xylose andD-glucose as carbon sources. Total solids content is 2% to 4% w./v. ofmedium. The medium is inoculated with 1% to 5% A. levanicum cells. Thecells are incubated for 12 to 24 hours at a temperature of 24 to 30 C.in shake flask culture in a Gyrotory shaker with a speed of 150 rpm. to300 rpm, at a pH value of 7-8. This produces 2.5-4.0x10 cells per ml. ofmedium.

A suitable medium for producing enzyme contains corn steep liquor oryeast extract as nitrogen sources and birchwood sulfite liquor andglucose as carbon sources, with a solids content of 5 to 10%. This isinoculated with 1 to 2% A. levanicum cells v./v. of medium. This isfermented for 18 to 24 hours at a pH value of 6 to 8 and a temperatureof 24 to 30 C. in a Gyrotory shaker at a speed of 150 rpm. to 300 r.p.m.This produces enzyme of 0.5 to 1.0 units of activity per ml. of medium.

The cells can be dried for 24 to 72 hours at a temperature of 20 to 30C., to a moisture content of 5 to 10%.

The cells can be sonified (ultrasonic fragmentation) to solubilize theenzyme at a force of to 300 watts for 3 to 15 minutes depending upon theamount to solubilize.

The foregoing are the criteria established when cell growth and enzymeinduction are not carried out simultaneously. Following are criteria forsimultaneous cell propagation and enzyme induction.

A suitable medium is yeast extract, corn steep liquor or yeast extractplus Bactopeptone, and D-xylose with a solids content of 2 to 4% Aninoculation of A. levanicum of 1 to 2% v./v. is added to the medium. Themicroa 3 organism is incubated for 24 to .40 hours at a temperature of24 to 30 C. and a pH of 5.5 to 9.0, in a Gyrotory shaker at a speed of100 r.p.m. to 300 r.m.p. This produces enzyme of potency of 0.3 to 0.4units per ml. of medium. These cells and enzymes can be dried or theenzymes separated as previously described.

Enzyme or cell-enzyme mixtures from any of the proceeding fermentationsmay be used to isomerize glucose to fructose according to the followingcriteria.

If air dried cells are used, from about 0.5% to about 1% w./v. of cellsare added to a glucose syrup of 36 to 50% glucose having a DB. (DextroseEquivalent) of 89 to 98 and a solids content of 40 to 60%. The glucosesyrup contains 0.1 to 0.6 nM Co++, 1 to 6 4M Mg++, and 10-40 ;:.Mphosphate. The pH is controlled between about 6.5 and about 7.5 and thetemperature is maintained between about 50 C. and about 60 C. Theconversion is allowed to proceed for about 24 to 72 hours.

The resultant product has 47 to 52% dextrose and 40 to 52% fructose.This syrup is treated by centrifugation to remove the cells followed byactivated carbon treatment and ion exchange to produce a commercialproduct of the following composition: 40-52% fructose, 47 to 52%glucose, 2 to 8% oligosaccharides, all on a dry substance basis.

Following are specific examples of the processes of this invention.

EXAMPLE NO. I

The process is carried out in a 250 ml. Erlenmeyer flask containing 100ml. of a suitable growth medium. The flask is shaken by a rotary shaker(200 revolutions/ minute with a 1 inch orbit) at a temperature of 23 C.for 24 hours. The flask is inoculated from a YM broth (pH 7.0) withAerobacter levanicum NRRL 13-1678. Two percent v./v. of A. levamcum isadded to the flask. The composition of the growth medium is thefollowing:

Percent Yeast extract 2.0

Glucose 0.2 Xylose 0.8 CoSO -7H 0.009 K01 0.18 Tap water 96.8

The pH is adjusted to 7.5 with a 50% KOH solution and the flaskcontaining the medium is sterilized for 10 minutes at 121 C. in anautoclave before being inoculated with A. levanicum'. After 24 hours thefermentation is stopped and the cells are harvested. 7.3 mg. cells/ml.growth medium are recovered and the enzyme recovered has 0.3 units/ml.activity based on the quantity of growth medium.

EXAMPLE NO. II

The process is carried out in thirty 1 liter Erlenmeyer flasks in atwo-stage process. The first stage is a growth process in which 300 ml.of medium is incubated at 2225 C. for 16 hours on a rotary shaker with a2 inch orbit at 175 r.p.m. The flasks are each inoculated with 3 ml. ofcultured Aerobacter levanicum. The composition of the growth medium isthe following:

Percent Yeast extract 2.0 Glucose 0.5 Tap water 97.5

Percent Birchwood sulfite liquor (as xylo'se) 4 CoS04-7H O 0.0036 KCl0.024 Tap Water 95.9724

The PH is adjusted t0'7.5 with 50% KOH.

The cells are harvested and washed by centrifugation and the resultingcell paste, 240 g., from 18 liters of total medium (both stages), may besonified to solubilize the enzyme, or the cells may be spread out andallowed to air dry at room temperature. In either procedure, the yieldof enzyme is 0.97 units per m1. of culture medium.

EXAMPLE NO. III

One gram of air dried cells having an activity of 480 units/ g. areadded to 100 ml. of 36% w./v. glucose solution containing in addition 610-' M C001 and 6x10 M MgSO The syrup conversion is allowed to continuefor 72 hours at 55 C. with the pH controlled automatically at 6.8 by theaddition of l N NaOH. The conversion is stopped by cehtrifugation. Thesyrup is purified by activated carbon treatment, ion exchanged andconcentrated. The resultant product has the following composition: 40%w./v. solids with 50.3% fructose and 49.7% glucose.

The identity of the conversion products as well as the percentcomposition of the syrups of the foregoing examples is confirmed 'byboth paper and thin layer chromatography and by gas chromatography ofthe silyl derivative by known methods as described in Journal of TheAssociation of Ofiicial Agricultural Chemists, 1198- 1201, 53 (b), 1970.

We claim:

1. A two-stage method of preparing an enzyme with substantial glucoseisomerizing activity from Aerobactcr levanicum comprising the steps of:

(a) combining in a first stage-culture medium, the microorganism,Aerobacter levanicum, and sources of nitrogen and carbon for a periodofabout 12 to about 24 hours, during which time predominately growth ofthe microorganism takes place, and

(b) in the second stage adding cobalt and unpurified hardwood sulfiteliquor to the said culture medium to induce the production of glucoseisomerizing enzyme, and

(c) thereafter recovering said glucose isomerizing enzyme. I

2. The method of claim 1 wherein the medium contains a magnesium salt.

3. The method of claim 2 wherein the yield of enzyme is at least about0.9 units per ml. of medium.

4. A method of converting glucose to fructose comprising treating anaqueous solution containing glucose with a glucose isomerizing enzymederived from the microorganism Aerobacter levdnicum as produced by theprocess of claim 1, maintaining the glucose isomerizing enzyme incontact with the glucose containing aqueous solution under glucoseisomerizing conditions, and con- 3,813,320 5 6 an enzyme concentrationof 1.0 units/ml. to 5.0 units/m1. OTHER REFERENCES for 24 to 72 hours. Tt 1 Sh k K h K k H k 7. The method of claim 6 wherein the conversion is3 3 enyus 0 en W 0 0k,

carried out with 0.5 to 1% w./v. dried cells as the enzyme Tsumura etaL: Biol. Chem., VOL 25, 616 9,

SOlll'CC. 19 1- 8. The method of claim 4 including the step of recover-5 Takasaki et ah: Perm by Perlman 561489, ing a converted glucosesolution having a composition of Acad Press 1969 47 to 52% glucose, 40to 52% fructose and 2 to 8% oligosaccharides on a dry substance basis.LOUIS MONACELL, Primary Examiner R f n e Cited 10 THOMAS G. WISEMAN,Assistant Examiner UNITED STATES PATENTS CL 3,616,221 10/1971 Takasakiet al 195-31 F 195-31 F

